Journal: Molecules

Article Title: Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model

doi: 10.3390/molecules29112676

Figure Lengend Snippet: SARS-CoV-2 Omicron BA.5 RBD expression and purification in E. coli . ( a ) Schematics of the sequence location of RBD in the SARS-CoV-2 spike protein. ( b ) Ribbon model of SARS-CoV-2 RBD with disulfide bond pairing. ( c ) SARS-CoV-2 Omicron BA.5 RBD expression and purification protocol in E. coli . ( d ) Binding of the SARS-CoV-2 Omicron BA.5 RBD to the hACE2 using an Octet-N1 Bio-Layer Interferometer. RBD was immobilized on a Ni-NTA sensor chip, and hACE2 was in the mobile phase.

Article Snippet: SARS-CoV-2 RBD (5 μg/mL) was immobilized on the Ni-NTA biosensor for 180 s, followed by an association phase with recombinant hACE2 (purity > 90% SDS-PAGE) (Bioworld Tech, St. Louis Park, MN, USA) diluted in kinetics buffer for 300 s. Dissociation was then carried out in the kinetics buffer for 300 s. Binding affinities were calculated using a 1:1 Langmuir binding model.

Techniques: Expressing, Purification, Sequencing, Binding Assay

Journal: Molecules

Article Title: Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model

doi: 10.3390/molecules29112676

Figure Lengend Snippet: hACE2 inhibition assay. ( a ) Steps of the hACE2 binding inhibition assay using bio-layer interferometry (BLI). RBD was immobilized on the biosensor chip, followed by antisera binding. hACE2 was loaded for the association and dissociation steps assessed in the kinetic buffer. ( b ) Enlarged figure of hACE2 association and dissociation step. ( c ) Inhibition of RBD binding to hACE2 by E. coli -expressed RBD-immunized antisera of one mouse from each group; “M” indicates the identity of the mouse.

Article Snippet: SARS-CoV-2 RBD (5 μg/mL) was immobilized on the Ni-NTA biosensor for 180 s, followed by an association phase with recombinant hACE2 (purity > 90% SDS-PAGE) (Bioworld Tech, St. Louis Park, MN, USA) diluted in kinetics buffer for 300 s. Dissociation was then carried out in the kinetics buffer for 300 s. Binding affinities were calculated using a 1:1 Langmuir binding model.

Techniques: Inhibition, Binding Assay

Journal: Frontiers in Microbiology

Article Title: Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

doi: 10.3389/fmicb.2023.1118025

Figure Lengend Snippet: The SARS-CoV-2 RBD binds to hACE2 via residues located on the three loops. (A) The structure of SARS-CoV-2 RBD and hACE2 complex. The RBM comprising the three loops (designated as RBML1 to RBML3) docks onto the surface of hACE2 (shown in purple; pdb entry 6LZG). (B) LigPlot+ plot of the interaction diagram. Hydrophobic contacts and hydrogen bonding between the two loops (RBML1 and RBML2) of the RBD and the two α-helices (α1 and α2) of hACE2 are shown at the top and the interactions between RBML3 and α1, α13, and the β-hairpin of hACE2 at the bottom. The horizontal dotted line represents the interface, in which the residues involved in direct intermolecular hydrophobic contacts are shown as semicircles with radiating spoke and linked by red dotted lines and hydrogens (<4 Å) are represented by green dashed lines.

Article Snippet: Recombinant hACE2 (Gln18-Ser740) was purchased from KMD Bioscience (Tianjin, China) which was expressed in HEK293 cells with >95% purity.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

doi: 10.3389/fmicb.2023.1118025

Figure Lengend Snippet: Structural and dynamics evidences for ACE2 binding origin. (A) Backbone-RMSDs of SARS-CoV-2 RBD and its deletion mutant shown as a function of time (left). Gyrate of proteins. SARS-CoV-2 RBD and its deletion mutant shown as a function of time (right). (B) ΔCα-RMSF data. SARS-CoV-2 RBDCtoS – SARS-CoV-2 RBD is marked in red and SARS-CoV-2 RBDC21_L3 – SARS-CoV-2 RBD in green (left). Conformational ensembles of RBML2 generated by MD simulations and shown by a “sausage” model with MOLMOL (right). (C) A 20-ns MD simulations showing structural dynamics of RBML2 in the apo state or ACE2-bound sate (left). Snapshots extracted from the MD trajectories at 10 and 15 ns, respectively, showing two distinct conformations in RBML2 (open and closed; middle). Comparison of the RBML2 RMSDs between SARS-CoV-2 RBD and the P491T mutant (right). (D) Structural mapping showing parallel molecular evolution removing steric hindrance and electric charge repulse present in the ancestral state. The clash occurs between Pro-502 of RBDs incapable of binding ACE2 and Lys-353 of ACE2, indicated by a cyan dashed circle, and the electric charge repulse between Asp-496 of the RBDs incapable of binding ACE2 and Asp-496 of ACE2, indicated by an orange dashed circle. In the RBD-hACE2 complex, hydrogen bonds are shown by yellow dashed lines and involved residues displayed as sticks.

Article Snippet: Recombinant hACE2 (Gln18-Ser740) was purchased from KMD Bioscience (Tianjin, China) which was expressed in HEK293 cells with >95% purity.

Techniques: Binding Assay, Mutagenesis, Generated, Comparison

Journal: Frontiers in Microbiology

Article Title: Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

doi: 10.3389/fmicb.2023.1118025

Figure Lengend Snippet: Purification, identification and functional characterization of recombinant RBDs. (A) Purification of refolded BtRBD and BtRBD|GY by SEC. Inset: SDS-PAGE analysis of the purified products, marked by a red arrow. “t 6 to t 9 ” denote collection tubes in SEC and “M” denotes protein molecular weight standard. (B) HPLC-Q-TOF-MS determining the molecular mass of BtRBD and BtRBD|GY. (C) Sensorgrams of SARS-CoV-2 RBD binding to the ACE2-immobilized chip surface (left top). The 125 nM analyte concentration was analyzed in duplicate. The concentrations used were 1,000–15.625 nM with two-fold serial dilutions. Sensorgrams of BtRBD to the chip surface (left bottom). The concentrations used were 10,000–625 nM with two-fold serial dilutions. Sensorgrams of BtRBD|GY to the chip surface (right bottom). The concentrations used were 40,000, 10,000, and 2,500 nM. Inset, schematic diagram of SPR experiment, in which the ligand hACE2 was covalently immobilized onto CM5 via its amine groups and the analytes (RBDs) flowed over the surface.

Article Snippet: Recombinant hACE2 (Gln18-Ser740) was purchased from KMD Bioscience (Tianjin, China) which was expressed in HEK293 cells with >95% purity.

Techniques: Purification, Functional Assay, Recombinant, SDS Page, Molecular Weight, Binding Assay, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

doi: 10.3389/fmicb.2023.1118025

Figure Lengend Snippet: The structural basis of the RBML1 of SARS-CoV-2 RBD interacting with hACE2. (A) Gly-446 and Tyr-449 of the RBML1 (colored in cyan) interact with Gln-42 and Asp-38 of hACE2 via three hydrogen bonds (pdb entry 6LZG). In the model of BtRBD, its RBML1 far away from the interface is colored in purple. (B) The lifetimes of the hydrogen bonds during 100-ns MD simulations. The dashed line represents the length threshold (4 Å) of a hydrogen bond.

Article Snippet: Recombinant hACE2 (Gln18-Ser740) was purchased from KMD Bioscience (Tianjin, China) which was expressed in HEK293 cells with >95% purity.

Techniques:

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: The role of CTHRC1 in SG function. ( a ) Sweat test of WT and Cthrc1 −/− mice. Dashed boxes indicate sweaty areas of mice paw pads. ( b ) Quantification of sweat dots on the paw pads of WT and Cthrc1 −/− mice. Each dot in the statistical graph represents the number of dark dots on one hind paw from an individual mouse ( n = 10). ( c ) Light-sheet microscopy images showing the SGs and their surrounding microvascular networks of WT and Cthrc1 −/− mice. 3D reconstruction of light-sheet microscopy images was preformed using Imaris software (Scale bar: 100 μm). ( d ) Representative H&E images of SGs and their adjacent microvessels. Arrowheads highlight the location of microvessels (Scale bar: 100 μm, top row; Scale bar: 25 μm, bottom row). ( e ) Representative images of laser Doppler blood flow perfusion and corresponding BF of WT and Cthrc1 −/− mice. Dashed boxes are at the same location as the ROIs in BF and indicate the palm pads of the mice left hind paws. ( f ) Analysis of the blood flow of the ROIs of WT and Cthrc1 −/− mice ( n = 3). Results were normalized to the WT mice. Results are presented as the mean ± SD, * p < 0.05, * * * p < 0.001. CTHRC1 Collagen triple helix repeat containing-1 protein, Cthrc1 collagen triple helix repeat containing-1 gene, SG sweat gland, WT wild type, H&E hematoxylin and eosin, BF bright field, ROIs regions of interest, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Microscopy, Software, Standard Deviation

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: The role of CTHRC1 in the development of SGs and their adjacent vasculature. ( a ) Comparison of SG markers K14 and K18 and secretion-related marker ATP1a1 between WT and Cthrc1 −/− mice. K14, K18 and ATP1a1, green; CD31, red; DAPI, blue (Scale bar: 50 μm). ( b – d ) The relative expression values of K8 , K18 and Atp1a1 mRNA of SGs of WT and Cthrc1 −/− mice ( n = 3). ( e ) Comparison of vasculature formation around SGs by staining for endothelial adherens (VE-cadherin) and tight junctions’ marker (ZO-1). VE-Cadherin and ZO-1, green; K18 and K19, red; DAPI, blue (Scale bar: 50 μm). ( f ) Scatter plot showing the down-regulated genes in the Cthrc1 −/− mice compared to WT mice by GO enrichment analysis (top 20 GO terms). The rich factor represents the ratio of the down-regulated genes to all genes enriched in the corresponding GO term. Dot size represents the number of down-regulated genes enriched in a specific GO term. Dot color represents the P value obtained by GO analysis. P < 0.05 was used to indicate significant enrichment. ( g ) The relative expression values of Agtr1a, Flt4 and Vegfd mRNA in dermal tissue of WT and Cthrc1 −/− mice ( n = 3). Results are presented as the mean ± SD, * * * p < 0.001, * * * * p < 0.0001; ns, not significant. CTHRC1 Collagen triple helix repeat containing-1 protein, Cthrc1 collagen triple helix repeat containing-1 gene, SG sweat gland, K cytokeratin, ATP1a1 ATPase Na + /K + transporting subunit alpha 1, CD31 platelet and endothelial cell adhesion molecule 1, WT wild type, mRNA messenger RNA, VE-cadherin vascular endothelial cadherin, ZO-1 zonula occludens-1, DAPI 4′,6-diamidino-2-phenylindole, GO gene ontology, MAPK mitogen-activated protein kinase, Agtr1a , angiotensin II receptor type 1a, Flt4 fms related receptor tyrosine kinase 4, Vegfd vascular endothelial growth factor D, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Comparison, Marker, Expressing, Staining, Standard Deviation

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: Identification of WT and Cthrc1 −/− mice-derived DMECs. ( a ) Schematic overview of the procedure carried out to isolate and purify DMECs. ( b ) Morphological properties of DMECs at days 5 and 7 (Scale bar: 100 μm, top row; Scale bar: 500 μm, bottom row). ( c ) Factor VII flow cytometry analysis of the positive rate of DMECs. DMECs displaying positive staining for ( d ) CD31 (Scale bar: 50 μm), ( e ) factor VII (Scale bar: 50 μm) and ( f ) BSL (Scale bar: 50 μm). ( g ) The relative expression values of Cthrc1 mRNA in DMECs ( n = 3). ( h ) Western blot analysis showing the CTHRC1 expression level of DMECs ( n = 3). Results are presented as the mean ± SD, * * * p < 0.001. Cthrc1 Collagen triple helix repeat containing-1 gene, WT wild type, DMECs dermal microvascular endothelial cells, CD31 platelet and endothelial cell adhesion molecule 1, BSL Bandeiraea simplicifolia lectin, mRNA messenger RNA, CTHRC1 collagen triple helix repeat containing-1 protein, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Derivative Assay, Flow Cytometry, Staining, Expressing, Western Blot, Standard Deviation

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: Regulation of CTHRC1 of the angiogenic ability of DMECs derived from WT and Cthrc1 −/− mice. ( a ) Immunofluorescence analysis of Ki67 expression of DEMCs after treatment with different concentrations of rmCTHRC1. The addition of VEGF (50 ng/ml) was used as a positive control. Ki67, green; DAPI, blue (Scale bar: 50 μm). ( b ) Quantification of the rate of Ki67 + DMECs in randomly acquired confocal images ( n = 5). ( c ) Florescent imaging displaying DiI-Ac-LDL uptake by DMECs in response to different concentrations of rmCTHRC1 stimuli. The addition of VEGF (50 ng/ml) was used as a positive control. DiI-AC-LDL, red; DAPI, blue (Scale bar: 50 μm). ( d ) Quantify the geometric mean fluorescence intensity of DiI-AC-LDL uptake by DMECs in randomly acquired confocal images ( n = 5). ( e ) Phase-contrast images of the capillary-like structures of DMECs treated with rmCTHRC1 (300 ng/ml) after 10 h. The addition of VEGF (50 ng/ml) and PBS was used as a positive and negative controls, respectively (Scale bar: 200 μm). ( f , g ) Quantification of tube formation degree of DMECs using tube length and nodes number (n = 9). ( h ) Immunofluorescence analysis of intercellular junction formation of DMECs treated with rmCTHRC1 (300 ng/ml) after 3 days (Scale bar: 100 μm). Results are presented as the mean ± SD, * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001; ns, not significant. CTHRC1 Collagen triple helix repeat containing-1 protein, Cthrc1 collagen triple helix repeat containing-1 gene, WT wild type, DMECs dermal microvascular endothelial cells, Ki67 antigen identified by monoclonal antibody Ki 67, rmCTHRC1 mouse CTHRC1 recombinant protein, VEGF vascular endothelial growth factor, DAPI 4′,6-diamidino-2-phenylindole, DiI-Ac-LDL DiI-labeled acetylated low-density lipoprotein, PBS phosphate buffered saline, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Derivative Assay, Immunofluorescence, Expressing, Positive Control, Imaging, Fluorescence, Recombinant, Labeling, Saline, Standard Deviation

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: rmCTHRC1 promotes the recovery of SG function by inducing adjacent microvascular network remodeling. ( a ) Workflow for rmCTHRC1 injection and evaluating the blood flow perfusion and SG function recovery of Cthrc1 −/− mice. ( b ) Representative images of laser Doppler blood flow and corresponding BF of Cthrc1 −/− mice treated with rmCTHRC1 (600 ng/ml) for 0, 3 and 7 days. The injection of PBS was used as a negative control. Dashed boxes are at the same location as the ROIs in BF and indicate the palm pads of the mice left hind paws. ( c ) Analysis of the blood flow of the ROIs ( n = 3). Results were normalized to the PBS group at 0 days. ( d ) Sweat test of Cthrc1 −/− mice treated with rmCTHRC1 for 7 days. The PBS group was used as a control. Dashed boxes indicate sweaty areas of mouse paw pads (at the same location as the ROIs in BF). ( e ) Quantification of sweat dots on the paw pads of Cthrc1 −/− mice. Each dot represents the number of dark dots on one hind paw from an individual mouse ( n = 5). ( f , g ) Representative H&E and immunofluorescence images of SGs of Cthrc1 −/− mice treated with rmCTHRC1 or PBS for 7 days (Scale bar: 100 μm, top rows; Scale bar: 50 μm, bottom rows). ( h , i ) Immunofluorescence analysis of the number (CD31), endothelium junction (VE-cadherin), and basement membrane (laminin) of SG adjacent microvessels. K18, ATP1a1, VE-cadherin, green; ZO-1, CD31, K19, Laminin, red; DAPI, blue (Scale bar: 100 μm, top rows; Scale bar: 50 μm, bottom rows). Results are presented as the mean ± SD, * p < 0.05. rmCTHRC1 Mouse CTHRC1 recombinant protein, SG sweat gland, Cthrc1 collagen triple helix repeat containing-1 gene, BF bright field, PBS phosphate-buffered saline, ROIs regions of interest, H&E hematoxylin and eosin, CD31 platelet and endothelial cell adhesion molecule 1, VE-cadherin vascular endothelial cadherin, K cytokeratin, ATP1a1 ATPase Na + /K + transporting subunit alpha 1, ZO-1 zonula occludens-1, DAPI 4′,6-diamidino-2-phenylindole, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Injection, Negative Control, Control, Immunofluorescence, Membrane, Recombinant, Saline, Standard Deviation